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P001. Activation of toll-like (TLR) and nod-like (NLR) receptors of human intestinal mucosal endothelial cells induces CEACAM1-mediated angiogenesis

A. Schirbel1,2, F. Rieder2, M. Phillips2, G. West2, C. Fiocchi2

1Charité Campus Virchow Klinikum, Berlin, Germany; 2Pathobiology and Gastroenterology, Digestive Disease Institute, Cleveland Clinic Foundation, Lerner Research Institute, Cleveland, OH, United States

Background and aim: CEACAM1 (carcinoembryonic antigen related cell adhesion molecule 1), a receptor for pathogenic bacteria is involved in adhesion, but also a potent mediator of angiogenesis. Human intestinal microvascular endothelial cells (HIMEC) get activated by bacterial products. Therefore we investigated whether TLR or NLR ligands (L) would induce HIMEC to express CEACAM1 and thus allow the homophilic binding of soluble CEACAM1 to further promote angiogenesis.

Material and methods: Normal and IBD HIMEC were exposed to optimal doses of FSL-1 (TLR2/6L), ultrapure LPS (TLR4L), iE-DAP (Nod1L) and MDP (Nod2L), the pro-angiogenic factors VEGF and bFGF serving as positive controls. CEACAM1 expression was assessed by immunoblotting and immunocytochemistry. Soluble CEACAM1 released in response to NLR/TLR activation was measured by ELISA. The pro-angiogenic action of recombinant (r)CEACAM1 was investigated by HIMEC proliferation, scratch wound migration, transmigration, matrigel and ex vivo aortic ring assay. Blocking experiments were performed with anti-CEACAM1.

Results: CEACAM1 expression was upregulated by all tested bacterial ligands with strongest effect of TLR2/6L and Nod2L.

Immunocytochemical staining of HIMEC monolayers revealed a distinct induction of CEACAM1, with TLR2/6 and Nod2 activation inducing predominantly cell surface expression, while TLR4 activation increased mainly the cytoplasmic form of CEACAM1.

In contrast, soluble CEACAM1 was released in a time-dependent manner primarily by stimulation with TLR4L and Nod2L.

Incubation with rCEACAM1 induced a small degree of HIMEC proliferation, while migration and transmigration were significantly (p = 0.04–0.007) increased in a dose-dependent fashion.

In the matrigel tube formation assay rCEACAM1 induced tube formation, while anti-CEACAM1 inhibited spontaneous and Nod1L-induced tube formation.

Incubation of murine aortic rings with rCEACAM1 significantly stimulated vessel sprouting compared to unstimulated aortic rings, whereas anti-CEACAM1 clearly inhibited TLR4L and Nod1L-induced vessel sprouting.

Co-stimulation with TLR2/6L or Nod2L and anti-CEACAM1 abolished the effect of both, TLR and NLR ligands completely. The effect of rCEACAM1 was inhibited by anti-CEACAM1, whereas control IgG had no effect.

Conclusions: TLR2/6 and Nod2 ligands preferentially induce the cell surface expression of CEACAM1 in HIMEC, whereas TLR4 and Nod2 ligands induce the release of the soluble form of CEACAM1. Furthermore, rCEACAM1 induces pro-angiogenic responses in HIMEC indicating that soluble CEACAM1 can also promote HIMEC angiogenesis. The effect of bacterial ligands can be abolished by blocking CEACAM1, demonstrating the role of CEACAM1 during TLR/NLR-mediated activation of endothelial cells.

These results show that expression of the pro-angiogenic molecule CEACAM1 is directly but differentially regulated by distinct bacterial products; promoting the release of soluble CEACAM1, which can in turn bind to cell surface CEACAM1 and further promote angiogenesis in an autologous fashion.

These novel findings suggest a broad regulatory role of various bacterial products in modulation of intestinal angiogenesis, and support a link between innate immunity and angiogenesis in IBD pathogenesis.